In the post-genomic era the field of proteomics has grown dramatically. For a multitude of applications, ranging from mass spectrometry analysis to high-content imaging, affinity-based assays are indispensable. Affinity-based assays rely on the detection of protein-protein-interaction (PPI) between a receptor and a ligand, such as an antibody and an antigen. In the case of the monitoring of an antigen-antibody-interaction the assay is also called an immunoaffinity assay. Immunoaffinity assays are the method of choice for testing the identity, quantity, and/or location of a polypeptide of interest. However, there are cases in which no suitable antibody is available. This disadvantage of immunoaffinity-based assays can be overcome by a method called “epitope tagging”, wherein a protein is tagged with an epitope, i.e. the binding part of an antigenic protein.
Epitope tagging is a technique in which a known epitope is fused to a recombinant protein by means of genetic engineering. By choosing an epitope for which an antibody is available, the technique allows the detection of proteins for which no antibody is available. Since the late 1980s epitope tagging has become a standard molecular genetics method for enabling rapid and effective characterization, purification, and in vivo localization of the protein products of cloned genes.
In the early days of proteomics the first commercially available tags were originally designed for protein purification. Examples of these early tags are FLAG, 6×HIS and the glutathione-S-transferase (GST) system. The 6×HIS tag relies on metal affinity and the GST system relies on affinity of GST to glutathione. FLAG is one of the first epitope tags used commercially.
Later on, the discovery of fluorescent protein reporters such as green fluorescent protein (GFP) made it possible to detect proteins intracellularly without the need of a secondary reagent. In this case, proteins of interest were tagged with the full-length protein sequence of GFP, rendering the tagged protein of interest fluorescent.
However, the problem with tags comprising full-length proteins such as GST maltose-binding protein (MBP) or GFP, is that they sometimes sterically interfere with subcellular protein localization or folding, which may compromise or abrogate the native function of the protein to be analyzed.
Therefore numerous small peptide-based epitope-tags such as c-myc, V5, HA, CBP or FLAG have been developed. Such tags have either a synthetic origin (FLAG) or are derived from viral (HA, V5) or endogenous mammalian (c-myc, CBP) proteins. They are characterized by a size of 8-26 amino acid residues and are detected by classical IgGs (poly- or monoclonal). One problem with tags derived from endogenous proteins is the fact that the tag-specific antibody generally also binds to the endogenous protein. If the interaction between the tag and the antibody is not specific, the assay may give false positives. Due to the competition between tagged protein and endogenous protein as binding partners for the antibody, the assay will be less efficient.
Although many immunoaffinity capture systems are available using tag-specific antibodies, there are still severe problems due to low affinity binding, unspecific interactions, batch to batch variations or reduced functionality of the antibodies upon covalent coupling to solid surfaces. Furthermore, a specific problem of known immunoaffinity detection and/or capture systems is their dependence on conventional antibodies evolved by the vertebrate immune system to detect the epitope-tagged protein.